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And Below-Ground Microbial Interactions with Arabidopsis

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And Below-Ground Microbial Interactions with Arabidopsis Impacts of sub-ambient and elevated CO2 on above- and below-ground microbial interactions with Arabidopsis. Alex Williams Department of Animal and Plant Science, The University of Sheffield Thesis submitted for the degree of Doctor of Philosophy Submitted: December 2017 This work is dedicated to Pauline Williams. The most beautiful life I knew. iii Abstract Over recent years, an increasing body of evidence has suggested that elevated atmospheric CO2 concentrations can alter plant microbial interactions. However, there is limited consensus whether these impacts will be positive or negative for plants in terms of disease resistance. Accordingly, there is a pressing need to gain a better understanding of the molecular and physiological mechanisms by which CO2 shapes the plant’s ability to interact with its biotic environment, which is essential to predict impacts of future climate scenarios on crop production. The work described in this thesis has used a range of CO2 concentrations, from past through present to future predicted concentrations, to study the immune response of the model plant Arabidopsis thaliana to aboveground pathogens and belowground rhizosphere bacteria. Furthermore, a novel developmental correction was established, which enables assessing the direct immunological effects of CO2 on microbial interactions without bias from age-related resistance arising from the stimulatory effects of CO2 on plant development. Changes in disease resistance at elevated CO2 (eCO2), against the necrotrophic fungus Plectosphaerella cucumerina (Pc) and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) were associated with changes in production and sensitivity of the phytohormones jasmonic acid (JA and salicylic acid (SA), respectively. However, priming of SA-dependent defence was not the only mechanisms contributing to eCO2-induced resistance against Hpa. The increased resistance to Hpa at sub-ambient CO2 (saCO2) against Hpa operated independently of SA signaling and was associated with changes in cellular redox state and priming of pathogen-inducible intracellular ROS. Based on the defence phenotypes of knock-down mutants in glycolate oxidase, the H2O2-generating enzyme of the photorespiration cycle, and transcriptional profiling of the peroxisomal catalase gene CAT2, it is proposed that the enhanced Hpa resistance at saCO2 is controlled by photorespiratory ROS. Below-ground, the root colonisation of a specialised rhizobacterial strain Pseudomonas simiae WCS417 was found to be dependent on CO2 concentration v and soil-nutritional status, whereas root colonisation by the soil saprophytic strain Pseudomonas putida KT2440 was largely unaffected by these variables. Hence, changes in atmospheric CO2 have a greater impact on specialist rhizosphere microbes. Furthermore, the ability of P. simiae WCS417 to promote plant growth and elicit an induced systemic resistance (ISR) was highly dependent on CO2 and nutritional status of the soil. These results suggest that the effects of atmospheric CO2 on rhizosphere microbes depend on the rhizosphere species in question and the nutritional status of the soil. To obtain a more global impression of the impacts of CO2 on rhizosphere interactions, rhizosphere soil was studied for bacterial community diversity and composition using PCR-based community profiling techniques. This revealed that CO2 has a measurable impact on microbial communities in a time-point dependent manner, whereby the effects of eCO2 are more pronounced at earlier stages and the effects of saCO2 are more pronounced at later stages. To study the biochemical basis of these CO2 effects on the rhizosphere effect, a new mass spectrometry-based method was developed to study quantitative and qualitative impacts of CO2 on non-sterile rhizosphere chemistry. These experiments revealed that the diversity of chemical signals greatly increases with rising CO2 concentrations, and that saCO2 and eCO2 are associated with rhizosphere enrichment of different classes of chemicals. Together, the results presented in this thesis provide novel insights into the mechanisms by which plants have adapted to past CO2 climates, and the potential impacts by which future CO2 scenarios will affect interactions with hostile and beneficial microbes. Further research is required to explore the combined impacts of eCO2 and other environmental changes due to global climate change, such as elevated temperatures and drought. vi Table of Contents Abstract ............................................................................................................. v Abbreviations and nomenclature .................................................................... x List of tables and figures................................................................................ xii Chapter 1: General Introduction ...................................................................... 1 1.1 Introduction ................................................................................................ 1 1.2 Shifting global climate. ............................................................................... 2 1.2.1 The greenhouse effect and the importance of CO2. ............................. 2 1.2.2 Past CO2 concentrations. .................................................................... 3 1.2.3 Current and future CO2 projections. .................................................... 4 1.3 Plant perception, recognition and responses to pathogenic microbes. ...... 6 1.4 Elevated CO2 effects on plant pathogen interactions. ................................ 8 1.4.1 Impacts of eCO2 on plant physiology. .................................................. 8 1.4.2 A meta-analysis of the impacts of eCO2 on plant disease. .................. 9 1.4.3 CO2 and pre-invasive resistance. ...................................................... 10 1.4.4 CO2: an enhancer or suppressor of post-invasive defences? ............ 11 1.4.5 Host defence responses to sub-ambient CO2. .................................. 16 1.5 Interactions with below-ground beneficial microbes................................. 18 1.6 Effects of CO2 on belowground interactions in rhizosphere. .................... 21 1.6.1 Effects of CO2 on rhizosphere chemistry. .......................................... 21 1.6.2 Effects of eCO2 on rhizosphere microbes. ......................................... 23 1.6.2 Effects of saCO2 on rhizosphere microbes. ....................................... 24 1.7 Thesis outline. ......................................................................................... 24 Chapter 2: Materials and Methods ................................................................. 27 2.1 Reagents and chemicals. ..................................................................... 27 2.2 Plant cultivation and growth conditions. ................................................ 27 2.3 Experimental set-up of growth system for metabolite collection. .......... 27 2.4 Development measurements and correction. ....................................... 28 2.5 Plant developmental correction (DC). ................................................... 28 2.6 Pathogenicity assays using Hpa and Pc. .............................................. 28 2.7 RT-PCR quantification of pathogen DNA. ............................................ 31 2.8 In situ detection of reactive oxygen species. ........................................ 32 vii 2.9 Targeted quantification of hormones. ................................................... 32 2.10 Metabolite extraction for untargeted profiling...................................... 33 2.11 Untargeted metabolic profiling by UPLC-qTOF-MSE. ......................... 33 2.12 Data preparation of MS data for statistical analysis. ........................... 34 2.13 Soil inoculation with Pseudomonas simiae WCS417 and Pseudomonas putida KT2440. ........................................................................................... 38 2.14 Induced systemic resistance assays. ................................................. 39 2.15 Bacterial community profiling by terminal-restriction fragment length polymorphism analysis. .............................................................................. 39 2.16 Community diversity and composition. ............................................... 40 2.17 Analysis of dissimilarity in T-RFLP patterns between samples. .......... 41 Chapter 3: Mechanisms of glacial-to-future atmospheric CO2 effects on plant immunity (adapted from Williams et al. 2017) ................................... 43 3.1 Abstract ................................................................................................... 43 3.2 Introduction .............................................................................................. 44 3.3 Results..................................................................................................... 46 3.4 Discussion ............................................................................................... 53 3.5 Supporting figures ................................................................................... 57 Chapter 4: Impacts of glacial-to-future atmospheric CO2 on bacterial rhizosphere colonisation in relation to plant growth and systemic resistance responses. .................................................................................... 67 4.1 Abstract ................................................................................................... 67 4.2 Introduction .............................................................................................
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